In cultured adult mammalian cells, growth arrest reliably occurs following a finite number of cell divisions. Commonly referred to as the Hayflick limit, this proliferative limit acts as an intractable species- and tissue-specific upper boundary in cultured somatic cells, with a limit of approximately 50 cell divisions for human lung fibroblasts. As a result of this restriction of cellular expansion, it is difficult to create standardized cultured somatic cells for widespread use, necessitating the use of immortal cell or tumor lines. Despite their durability as cell sources, all immortalized cells contain genetic mutations allowing them to circumvent proliferative limits, creating doubt in their efficacy as representative tissues for primary pharmacological applications and other bioassays. Similarly, in injured or otherwise damaged tissues transplantation of healthy and/or genetically modified cells, is currently an attractive therapeutic possibility. However, the limited ability to controllably expand cell populations is a barrier to autologous re-transplantation approaches, and necessitates the use of donor tissue, creating ethical and immunological concerns.
The underlying mechanisms defining proliferative lifespan are believed to vary between species. In cultured rodent cells, the regulatory entities establishing maximal replicative lifespan are unclear, and may involve a mechanism for counting mitotic events and/or phenomena related to culture conditions. Strong evidence indicates that the replicative lifespan of cultured human cells is primarily regulated by the length of telomeres, repeated hexameric nucleotide repeats at each end of the chromosomes that are believed to protect against replication-associated damage and chromosome end-fusion. In most somatic cells, chromosomal telomeres are progressively shortened as a result of incomplete end replication, and cells cease to divide upon reaching critical telomere length. Telomerase, the ribonuclear-protein enzyme involved in de novo extension of telomeres, appears essential for maintaining replicative competency, as human cells engineered to express catalytic telomerase reverse transcriptase (TERT) have a dramatically increased replicative lifespan in culture.
Mammalian somatic cells are limited to a finite number of mitotic events before entering irreversible growth arrest. This limited replicative competency provides an inherent restriction on expansion of cell populations in culture, and may be associated with age-related infirmity and disease in a broad range of tissues.
There is therefore, an urgent need in the art to provide long-term cultures of non-immortalized cells for drug discovery, transplantation, diagnostics, therapeutics and related medical purposes.